Templated alkylation of hexahistidine with Baylis-Hillman esters

Article abstract of DOI:10.1039/c3cc43271h

Alkylation of one of the imidazole rings of hexahistidine with Baylis-Hillman esters tethered to nitrilotriacetate residue was achieved in aqueous solutions at neutral pH and at micromolar concentrations in the presence of Ni²⁺, Cu²⁺, or Zn²⁺ cations. The utility of the approach for selective functionalization of His-tagged recombinant proteins was demonstrated by attachment of a fluorescent label to recombinant protein A with an alkynyl group followed by a "click" 1,3-dipolar cycloaddition reaction.

Full text of DOI:10.1039/c3cc43271h

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Templated alkylation of hexahistidine with
Baylis–Hillman esters†
Cite this: Chem. Commun., 2013,
49, 9042
Xing Xin Liu and Artem Melman*
Received 2nd May 2013,
Accepted 8th August 2013
DOI: 10.1039/c3cc43271h
www.rsc.org/chemcomm
Alkylation of one of the imidazole rings of hexahistidine with Baylis–
Separation of proteins expressed by bacteria from recombinant
Hillman esters tethered to nitrilotriacetate residue was achieved in DNA vectors and native proteins is commonly done by affinity
aqueous solutions at neutral pH and at micromolar concentrations in chromatography through encoding of a hexahistidine motif at the
the presence of Ni2+, Cu2+, or Zn2+ cations. The utility of the approach beginning or at the end of an amino acid sequence of a recombinant
for selective functionalization of His-tagged recombinant proteins was protein.16 This method became the standard technology for prepara-
demonstrated by attachment of a fluorescent label to recombinant tion of recombinant proteins. The hexahistidine tag constitutes an
protein A with an alkynyl group followed by a ‘‘click’’ 1,3-dipolar excellent opportunity toward selective chemical covalent derivatization
cycloaddition reaction.
of these proteins. Such an approach can utilize the ability of the
hexahistidine sequence to produce a metal complex that can serve as
Chemical functionalization of proteins is a common approach a template for subsequent intramolecular formation of a covalent
toward their utilization in a plethora of functional assemblies. It bond with one of the histidine residues of the sequence. In many
is extensively used in the study of protein structure,1 labelling of aspects this approach resembles the methodology of temporary
proteins,2 their immobilization,3 drug targeting,4 modification of tethering of two reactants through a covalent17 or a coordinative18
immunological properties,5 bioanalytics,6 and other applications.7 bond, which is followed by the intramolecular formation of a
Availability of recombinant proteins that can be expressed and permanent covalent bond and final removal of the temporary tether.
purified using standard methodologies makes essentially any
Although there were recent attempts to use formation of Ni2+
protein with a known amino acid sequence available through nitrilotriacetate (NTA) complexes for facilitating alkylation of histi-
routine protocols. In contrast, due to the presence of multiple dine residues in his-tagged proteins they suffered from low reactivity
residues of amino acids in any protein, conventional methods of imidazole rings towards conventional alkylation reactions.19 An
producing protein conjugates through reactions of side chains of alternative can be found in Baylis–Hillman esters that are versatile
cysteine8 and lysine9 rarely provide well-defined protein conjugates. reagents for alkylation of a variety of nucleophilic groups through a
To overcome this problem other approaches involving site-specific concerted addition–elimination process.20 Particularly interesting is
incorporation of unnatural amino acids,10 introduction of aldehyde reported21 high reactivity of Baylis–Hillman esters to imidazole in
tags into proteins by the formylglycine generating enzyme,11 and the presence of water, which can make these compounds suitable as
native chemical ligation12 were tried. To increase versatility, protein histidine-reacting agents. Here we report on successful alkylation of
bioconjugates are prepared in two stages through attachment of a hexahistidine using Baylis–Hillman esters templated by transition
reactive functionality that is capable of highly selective subsequent metal cations, and application of this methodology for derivatization
transformations such as biotinylation.13 Alternatively, proteins are of recombinant proteins (Fig. 1).
functionalized with groups capable of highly specific bioorthogonal
Our approach involved tethering of NTA functionality to the Baylis–
reactions14 such as cycloaddition or ‘‘click reactions’’ with comple- Hillman group through the ester group using a flexible linker. In that
mentary components attached to a small molecule or another case formation of the Ni2+ ternary complex between the hexahistidine
protein.15 However, preparation of well-defined conjugates from fragment and NTA would bring the reactive double bond of the Baylis–
an arbitrary protein remains a considerable challenge.
Hillman ester group into the vicinity of one of the imidazole rings that
do not form the coordination bond with Ni2+ cations. The resultant
addition–elimination process would form a C–N covalent bond while
breaking the linkage with NTA leaving the ArCHQCH(CO2Me)CH2–
moiety attached to the hexahistidine residue.
Clarkson University, Department of Chemistry & Biomolecular Science, Potsdam,
New York, USA. E-mail: amelman@clarkson.edu; Fax: +1 315-268-6610;
Tel: +1 315-268-4405
† Electronic supplementary information (ESI) available: Syntheses of compounds
6, 7a,b and 14, ESI-MS and NMR spectra, images of SDS-PAGE. See DOI: 10.1039/
c3cc43271h
Syntheses of NTA tethered Baylis–Hillman esters were done
through conversion of aldehydes 1a,b into Baylis–Hillman adducts
c
9042 Chem. Commun., 2013, 49, 9042--9044
This journal is The Royal Society of Chemistry 2013

View Article Online
Communication
ChemComm
Fig. 1 General approach to alkylation of an imidazole residue in the hexa-
histidine tag through formation of a ternary metal complex with NTA function
carrying a Baylis–Hillman ester alkylating moiety.
Fig. 3 Above: conversion in alkylation of N-acetylhexahistidine 9 with ester 7a
at different concentrations of N-methylimidazole 8 after 16 h. Below: schematic
representation of transition of binding of Ni2+ by N-acetylhexahistidine 9 from
the tridentate mode in ternary complex 11a to the bidentate mode in the
quaternary complex 11b induced by N-methylimidazole (8).
with addition of 1 eq. of nickel salt to the reaction mixture provided
only a marginal improvement of the conversion (Fig. 3). The observed
low rates of the reaction were attributed to the formation of a rigid
ternary complex of type 11a through tridentate binding of Ni2+ cations
by N-acetylhexahistidine. In such a complex, Ni2+ bound imidazole
rings can sterically shield remaining non-coordinating imidazole rings
from approaching the alkylating Baylis–Hillman ester group.
To overcome this shielding we added an additional monodentate
ligand capable of binding Ni2+ cations to the reaction mixture to
force switching of coordination of Ni2+ by N-acetylhexahistidine to a
bidentate mode through formation of a quaternary complex of type
11b. Indeed, addition of imidazole or N-methylimidazole 8 to the
reaction mixture resulted in dramatic increase in the conversion.
The maximal conversion of 30% in 16 h was achieved at 8 eq. of
N-methylimidazole. Further increase of concentration of N-methyl-
imidazole decreases reaction rates due to its competition with
N-acetylhexahistidine for Ni2+ binding.
Further study showed that this templated alkylation can be
further accelerated by replacing Ni2+ cations with Cu2+, and Zn2+
in the presence of 6 eq. of N-methylimidazole 8 (Fig. 4). Further
increase in conversion above 85% is possible through using two
equivalents of alkylating agent 7a or by increase of reaction time.
However, under these conditions dialkylation of N-acetylhexa-
histidine with two molecules of ester 7a was observed as evidenced
by formation of a M À H+ peak at 1229.
Scheme 1 Synthesis of NTA tethered Baylis–Hillman esters 7a,b.
2a,b followed by their reaction with acyl chloride prepared
from glutaric anhydride 3 through successive treatment with
N-hydroxysuccinimide/DMAP and thionyl chloride. Reaction of
the resultant active ester 5a,b with L-lysine derived amine 6
provided the desired esters 7a,b (Scheme 1).
The approach was initially studied on a model reaction of ester 7a
(0.25 mM), N-acetylhexahistidine (0.25 mM), and Ni(OAc)2 (0.25 mM)
in a mixture of methanol–water. To determine the contribution of an
intermolecular alkylation of N-acetylhexahistidine 9 with ester 7a a
control experiment was conducted, which excluded Ni(OAc)2. The
reaction was analyzed by a direct injection into an ESI-TOF spectro-
meter (negative mode) after 16 h, and concentrations of N-acetyl-
hexahistidine 9 and expected alkylation products of type 10
were estimated from relative intensities of M À H+ peaks at
881 D and 1055 D (Fig. 2). The validity of ESI-MS for monitoring
of the conversion was proved by comparison of a set of parallel
experiments using analysis of the reaction mixture by HPLC.
The control experiment in the absence of Ni2+ showed only minor
(o3%) amounts of alkylation products of type 10. Initial experiments
Fig. 4 Comparison of conversion alkylation of N-acetylhexahistidine 9 (250 mM)
with ester 7a (250 mM) templated by formation of their mixed Ni2+, Cu2+ or Zn2+
(250 mM) complexes in the presence of 8 after 16 h.
Fig. 2 Alkylation of N-acetylhexahistidine 9 with ester 7a templated by for-
mation of their ternary complex with Ni2+ cations.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 9042--9044 9043

View Article Online
ChemComm
Communication
monodentate ligand such as N-methylimidazole, and a metal
cation such as Ni2+, Cu2+, or Zn2+. While currently achieved
alkylation time is higher than in the existing bioconjugation
methods, this reaction can be a promising way for specific
derivatization of recombinant proteins possessing a hexahistidine
tail through introduction of an alkynyl function that can be used
for subsequent bioorthogonal cycloaddition reactions. Studies of
this versatile approach are currently being extended to develop-
ment of new alkylating agents to perform derivatizations of
recombinant proteins with quantitative yield using stoichiometric
amounts of reagents at low micromolar concentrations in vitro
and in vivo.
This work was supported by National Science Foundation
grant CHE 1150768.
Fig. 5 Derivatizations of protein A 12 with an alkynyl function through the
alkylation with ester 7b followed by a ‘‘click’’ cycloaddition with fluorescein azide
14 to produce fluorescent protein 15. Left bottom: scan of the fluorescent image
of gel electrophoresis of modified protein A.
Notes and references
1 (a) W. L. Vos, R. B. M. Koehorst, R. B. Spruijt and M. A. Hemminga,
J. Biol. Chem., 2005, 280, 38522; (b) R. Roy, S. Hohng and T. Ha,
Nat. Methods, 2008, 5, 507.
2 (a) G. T. Hermanson, Bioconjugate techniques, Elsevier Academic
Press, Amsterdam, Boston, 2008; (b) L. Z. Kelman, V. Naktinis and
M. Odonnell, DNA Replication, Academic Press Inc, San Diego, 1995,
p. 430.
3 C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan and
R. Fernandez-Lafuente, Enzyme Microb. Technol., 2007, 40, 1451.
4 J. W. Park, K. L. Hong, D. B. Kirpotin, G. Colbern, R. Shalaby,
J. Baselga, Y. Shao, U. B. Nielsen, J. D. Marks, D. Moore,
D. Papahadjopoulos and C. C. Benz, Clin. Cancer Res., 2002, 8, 1172.
5 P. Caliceti and F. M. Veronese, Adv. Drug Delivery Rev., 2003,
55, 1261.
These results demonstrate the possibility of selective alkylation of
hexahistidine in aqueous solution at micromolar concentrations,
and suggest that this method can be used for selective derivatization
of recombinant proteins possessing a hexahistidine tag. The reac-
tion of alkylation can be used for introduction of a ‘‘sticky end’’
group such as alkynyl, which can be a versatile substrate for a
subsequent bioorthogonal reaction to introduce variety of function-
alities. To prove this approach a sample of recombinant protein A
possessing a hexahistidine tag was alkylated with alkynyl carrying
ester 7b to perform two stage derivatization with a fluorescent
functionality. On the first stage protein A (16 mM) was treated with
2 eq. of ester 7b, 12 eq. of N-methylimidazole, and 2 eq. of either
Cu2+ or Zn2+ cations to produce alkynyl functionalized protein 13. To
determine the rate of possible intermolecular alkylation of protein A
of the hexahistidine sequence, or of any other reaction site, a control
experiment in the absence of these metal cations was also per-
formed. After incubation for 24 hours all three reaction mixtures
were treated with diazidofluorescein 14 and TBTH/ascorbate for 5 h
followed by separation of reaction products using SDS-PAGE (Fig. 5).
Strong fluorescence of the attached residue allowed visual obser-
vation of the derivatized product 15. As can be seen in Fig. 5 both
reactions in the presence of Cu2+ and Zn2+ cations showed successful
derivatization of recombinant protein A. The otherwise identical
6 S. Cosnier, Biosens. Bioelectron., 1999, 14, 443.
7 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009,
48, 6974.
8 (a) T. P. King, Y. Li and L. Kochoumian, Biochemistry, 1978, 17, 1499;
(b) G. L. Ellman, Arch. Biochem. Biophys., 1959, 82, 70; (c) D. Macmillan,
R. M. Bill, K. A. Sage, D. Fern and S. L. Flitsch, Chem. Biol., 2001, 8, 133;
(d) G. Gorin and G. Doughty, Arch. Biochem. Biophys., 1968, 126, 547.
9 (a) P. D. Bragg and C. Hou, Arch. Biochem. Biophys., 1975, 167, 311;
(b) A. Rifai and S. S. Wong, J. Immunol. Methods, 1986, 94, 25;
(c) L. Peng, G. J. Calton and J. W. Burnett, Appl. Biochem. Biotechnol.,
1987, 14, 91; (d) J. M. McFarland and M. B. Francis, J. Am. Chem.
Soc., 2005, 127, 13490.
10 C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith and P. G. Schultz,
Science, 1989, 244, 182.
11 D. Rabuka, J. S. Rush, G. W. deHart, P. Wu and C. R. Bertozzi, Nat.
Protocols, 2012, 7, 1052.
12 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent, Science,
1994, 776.
13 M. Wilchek, E. A. Bayer and O. Livnah, Immunol. Lett., 2006, 103, 27.
control reaction in the absence of metal cations produced only trace 14 (a) J. C. Jewett and C. R. Bertozzi, Chem. Soc. Rev., 2010, 39, 1272;
(b) S. K. Mamidyala and M. G. Finn, Chem. Soc. Rev., 2010, 39, 1252.
15 (a) H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
amounts of protein A derivatized with the fluorescent label thus
indicating a predictably extremely slow rates of intermolecular
2001, 40, 2004; (b) R. Rossin, P. R. Verkerk, S. M. van den Bosch,
alkylation of protein A under the high dilution conditions employed.
The ratio of spot fluorescence intensities was 18.6 (Zn2+): 23.3 (Cu2+):
1 (no metal cation). Quantitative measurement of the dye/protein
ratio by UV-Vis absorption was found to be 35% for the reaction in
the presence of Zn2+, and 60% for the reaction with Cu2+. These
reaction rates were several times lower than reported alkylation of a
R. C. M. Vulders, I. Verel, J. Lub and M. S. Robillard, Angew. Chem.,
Int. Ed., 2010, 49, 3375; (c) S. T. Laughlin, J. M. Baskin, S. L. Amacher
and C. R. Bertozzi, Science, 2008, 320, 664.
16 (a) V. Gaberc-Porekar and V. Menart, Chem. Eng. Technol., 2005,
28, 1306; (b) E. Hochuli, W. Bannwarth, H. Dobeli, R. Gentz and
D. Stuber, Nat. Biotechnol., 1988, 6, 1321.
17 (a) M. D. Bachi and A. Melman, Synlett, 1996, 60; (b) M. D. Bachi,
Y. V. Bilokin and A. Melman, Tetrahedron Lett., 1998, 39, 3035.
protein containing a His10 tag,19a most likely due to substantially 18 (a) G. Stork and T. Y. Chan, J. Am. Chem. Soc., 1995, 117, 6595;
(b) F. Bertozzi, R. Olsson and T. Frejd, Org. Lett., 2000, 2, 1283.
19 (a) S. Uchinomiya, H. Nonaka, S. Fujishima, S. Tsukiji, A. Ojida and
higher flexibility of His10 over the commonly used hexahistidine tag.
We demonstrated that the hexahistidine group can be alkylated
I. Hamachi, Chem. Commun., 2009, 5880–5882; (b) M. Hintersteiner,
in aqueous solutions at micromolar concentrations of reagents
assembled from Baylis–Hillman esters tethered to nitrilotriacetate
through a flexible linker. The reaction is templated by forma-
T. Weidemann, T. Kimmerlin, N. Filiz, C. Buehler and M. Auer,
ChemBioChem, 2008, 9, 1391.
20 D. Basavaiah, B. S. Reddy and S. S. Badsara, Chem. Rev., 2010,
110, 5447.
tion of the quaternary complex composed of these reactants, a 21 H. Li, X. X. Wang and Y. M. Zhang, Tetrahedron Lett., 2005, 46, 5233.
c
9044 Chem. Commun., 2013, 49, 9042--9044
This journal is The Royal Society of Chemistry 2013